Nanostructured surfaces

ABSTRACT

The present invention is directed to methods for inhibiting growth of bacteria and to nanometer scale surfaces having antibacterial properties.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/322,366, filed on Apr. 9, 2010 and U.S. Provisional PatentApplication No. 61/418,838, filed on Dec. 1, 2010, each of which ishereby incorporated by reference in their entireties.

FIELD

The present invention relates to the field of nanotechnology and, inparticular, to surface geometries on the nanometer scale that are usefulto decrease bacterial adhesion. The present invention also relates tothe field of implants and/or medical devices having such surfacegeometries which decrease bacterial adhesion and thereby lower the riskof infection to patients into which the implant or medical device isplaced or implanted.

BACKGROUND

The attachment of bacteria to surfaces, sometimes referred to as biofilmformation, often occurs in two major steps. The first is an initialattachment of the bacteria to the surface and the second is acell-to-cell proliferation to form multilayered bacterial clusters. Theinitial attachment of the bacteria to the surface is thought to beaffected by surface roughness, surface charge, and hydrophobicity.Attachment of bacteria is undesirable on surfaces of devices that areintended to be placed in the body of an individual because of the riskof infection to the individual. Such devices include needles, tubes,implants, medical devices and the like. Attachment of bacteria isfurther undesirable on surfaces where materials or devices are preparedprior to insertion into an individual. Attachment of bacteria is alsoundesirable on surfaces where food preparation takes places, as food canbecome contaminated prior to ingestion. It is desirable to produce asurface where adherence and/or proliferation of bacteria is decreasedthereby decreasing the risk of infection.

It is therefore an object of the present invention to create a surfacecharacterized by at least one of surface roughness, surface chargeand/or hydrophobicity where the ability of bacteria to adhere to thesurface is decreased. It is a further object of the present invention toprovide a substrate surface with a surface geometry on the nanometerscale. It is a further object of the present invention to alter thesurface of a substrate in a manner to reduce bacterial adhesion and/orproliferation. These and other objects, features, and advantages of theinvention or certain embodiments of the invention will be apparent tothose skilled in the art from the following disclosure and descriptionof exemplary embodiments.

SUMMARY

Embodiments of the present invention are directed to surfaces ofsubstrates characterized by at least one of surface roughness, surfacecharge and/or hydrophobicity where the ability of bacteria to adhere,proliferate, and/or colonize to the surface is decreased, inhibitedand/or reduced. The surface is referred to herein as being“antibacterial” to the extent that the ability of bacteria to adhere tothe surface is decreased thereby reducing the proliferation of bacteriaand thereby reducing the risk of infection or illness due to thepresence of bacteria. It is to be understood that embodiments of thepresent invention allow some adherence of bacteria. However, the surfacecharacteristics of the present invention, such as a nanometer scalegeometry, reduce the ability of bacteria to adhere to the surface.Further embodiments of the present invention include surfacecharacteristics, such as a nanometer scale geometry, which reduce theability of bacteria to proliferate.

Embodiments of the present invention are also directed to use of asubstrate surface with a surface geometry on the nanometer scale toreduce proliferation of bacteria. Embodiments of the present inventionare further directed to methods of altering a surface of a substrate ina manner to reduce bacterial adhesion, proliferation and/ordifferentiation on the substrate surface. Embodiments of the presentinvention are further directed to methods of altering a surface of asubstrate to create a nanometer scale surface geometry and using thesubstrate to reduce bacterial proliferation.

Embodiments of the present invention are still further directed tomethods of reducing the risk of bacterial infection from the insertionor implantation of devices into an individual. Embodiments of thepresent invention are even still further directed to methods of reducingthe risk of bacterial infection from the use of surfaces that maytransmit bacteria during the processing of food or the manufacture ofdevices or materials intended to be inserted or implanted in anindividual. According to the methods, the presence of bacteria isreduced, for example when compared to a surface lacking the nanometerscale surface geometry, thereby reducing the risk of infection orillness due to the presence of bacteria.

According to certain aspects of the present invention, a substratesurface is provided that has a nanometer scale surface roughness. Asubstrate surface having a nanometer scale surface roughness possesses ahigher percentage of atoms at the substrate surface and/or increasedportions of surface defects and/or greater numbers of materialboundaries at the surface that are influencing protein interactionsimportant for controlling cell functions. A substrate having a nanometerscale surface roughness according to the present invention ischaracterized by the reduced adhesion and/or proliferation, and/ordifferentiation of bacterial cells on the surface compared to asubstrate lacking the nanometer scale surface roughness. In addition ananometer scale surface roughness according to the present disclosuredecreases inflammation because of an altered surface energy whichpromotes the adsorption of proteins, such as vitronection andfibronection, that decreases inflammatory cell functions. In addition, ananometer scale surface roughness according to the present disclosuredecreases bacterial functions because of an altered surface energy whichpromotes the adsorption of proteins, such as vitronection andfibronection, that decreases bacterial functions. In addition ananometer scale surface roughness according to the present disclosureincreases bone formation and growth because of an altered surface energywhich promotes the adsorption of proteins, such as vitronection andfibronection, that promote bone cell functions. The substrate of thepresent invention may also exhibit a surface charge and/orhydrophobicity where the ability of bacteria to adhere to the surface isdecreased compared to a surface having a different surface charge and/orhydrophobicity. Accordingly, substrate surfaces of the present inventionare useful to reduce the risk of bacterial infection when the substrateis inserted or implanted into an individual. Accordingly, aspects of thepresent invention contemplate methods of reducing bacterialproliferation, and therefore reducing the risk of bacterial infection orillness, by providing a surface having a nanometer scale surfaceroughness and/or desirable surface charge and/or desirablehydrophobicity for insertion or implantation into an individual.According to certain aspects of the present invention, a method isprovided whereby a substrate surface is altered to create a nanometerscale surface roughness, and then the substrate is inserted or implantedinto an individual. According to this aspect, a method is provided toreduce bacterial adhesion, proliferation or differentiation on thesurface of the substrate by altering the substrate surface to include ananometer scale surface roughness and thereby also reduce the risk ofbacterial infection or illness when the substrate is introduced orimplanted into an individual.

According to particular aspects of the present invention, a method isprovided of altering the surface of a substrate by contacting thesurface of the substrate with a nano-roughing agent. A nano-roughingagent according to the present disclosure produces a nano-rough surfaceon the substrate. A nano-rough surface is characterized by a surfacemorphology having structural features with nanometer dimensions.

According to particular aspects of the present invention, a method isprovided of altering the surface of a substrate by contacting thesurface of the substrate with a solution of a nano-roughing agent, suchas a bacterial lipase and etching the surface of the substrate by thebacterial lipase. According to certain aspects, the bacterial lipase isproduced by Rhisopus arrhisus or Candida cilindracea. According tocertain other aspects, the surface of the substrate is contacted with asolution of Rhisopus arrhisus or Candida cilindracea. According to stillother aspects, the etching of the surface with nano-roughing agentproduces a nanometer scale surface geometry.

According to other aspects, a method is provided of inhibiting growth ofbacteria on the surface of a substrate by providing the surface of thesubstrate with a nanometer scale surface geometry, contacting thesurface with bacteria, and inhibiting adherence and/or growth of thebacteria to the surface. For purposes of the embodiments of the presentinvention, the step of contacting the surface with bacteria includesbacteria coming into contact with the substrate as would be common whenbacteria is transmitted through direct touch or indirect methods such asairborne travel. According to this aspect, bacterial infection in ananimal including a human, including the risk of bacterial infection, isreduced when the substrate with the nanostructured surface, such as anendotracheal tube made of PVC or silicone or other like material, isinserted into the animal.

A still further embodiment is provided for a method of reducing growthof bacteria on a surface of a substrate by altering the surface of thesubstrate to produce a nanometer scale surface geometry by contactingthe surface of the substrate with a solution of a nano-roughing agentsuch as a bacterial lipase and etching the surface of the substrate bythe bacterial lipase to produce the nanometer scale surface geometry.According to one embodiment, the bacterial lipase is produced byRhisopus arrhisus or Candida cilindracea. According to anotherembodiment, the surface of the substrate is contacted with a solution ofRhisopus arrhisus or Candida cilindracea. The substrate, such as anendotracheal tube, may be formed from PVC or silicone or other likematerials susceptible to etching by bacterial lipases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict scanning electron microscope (SEM) images ofuntreated PVC samples at magnifications of 1 k and 42 k.

FIGS. 2A and 2B depict SEM images at magnification of 20 k of PVCtreated after 24 hours with 0.5% solution of R. arrhisus and 0.5%solution of C. cilindracea.

FIGS. 3A and 3B depict SEM images of PVC treated with 0.1% C.cilindracea solution after 48 hrs at magnification of 5 k and 42 k.

FIGS. 4A, 4B, 4C and 4D depict SEM images of PVC treated after 48 hrswith 0.1% R. arrhisus solution at magnification of 10 k and 42 k andwith 0.5% R. arrhisus solution at magnification of 20 k and 42 k.

FIG. 5 is a graph depicting bacteria growth of Staphylococcus aureus onuntreated PVC (blue bar), PVC treated with C. cilindracea (green bar)and PVC treated with R. arrhisus (tan bar).

FIGS. 6A and 6B depict SEM images of untreated PVC treated (20 kmagnification) and PVC treated with R. arrhisus solution (20 kmagnification).

FIG. 7 is a graph of macrophage density for polypropylene substratesthat are untreated, treated with isoamyl acetate, treated withdichloromethane, treated with isoamylacetate and zinc chloride andtreated with dichloromethane and zinc. Nanostructured polypropylene(without LPS) surfaces had less macrophage density compared to theuntreated surface.

FIG. 8 is a graph of macrophage density for polypropylene substratesthat are untreated, treated with isoamyl acetate, treated withdichloromethane, treated with isoamylacetate and zinc chloride andtreated with dichloromethane and zinc. Nanostructured polypropylene(with LPS) surfaces had less macrophage density compared to theuntreated surface.

FIGS. 9A and 9B are images showing the relative amount of bacteria on anuntreated polyethyl ethyl ketone substrate and a polyethyl ethyl ketonesubstrate with a nano-rough or nano-structured surface. The polyethylethyl ketone substrate with a nano-rough or nano-structured surface hadlower amounts of Staphylococcus epidermis bacteria.

FIG. 10 is a graph depicting relative amounts of Staphylococcus aureusbacteria for an untreated polyvinyl chloride substrate from anendotracheal tube, a polyvinyl chloride substrate from an endotrachealtube treated with Candida cilindracea (Nano-C) and a polyvinyl chloridesubstrate from an endotracheal tube treated with Rhisopus arrhisus(Nano-R) to create nano-scale features. The polyvinyl chloride with anano-rough or nano-structured surface had lower amounts ofStaphylococcus aureus bacteria.

FIG. 11 is a graph of Staphylococcus epidermis colonization forpolypropylene substrates that are untreated, treated with isoamylacetate, treated with dichloromethane, treated with isoamylacetate andzinc chloride and treated with dichloromethane and zinc. Nanostructuredpolypropylene had less Staphylococcus epidermis colonization compared tothe untreated surface.

FIG. 12 is a graph of calcium deposition in micrograms/cm² for untreatedpolyethyl ethyl ketone (PEEK), treated polyetherether ketone(nanoPEEK1), treated polyetherether ketone (nanoPEEK2) and glass. Thetreated polyetherether ketone had enhanced osteoblast calcium depositioncompared to untreated polyetherether ketone and osteoblast calciumdeposition similar to glass.

FIGS. 13A and 13B are images of the surface morphology of untreatedpolyetherether ketone and nano-structured polyetherether ketone.

FIGS. 14A and 14B are images of the surface morphology of untreatedpolyglycolic acid and nano-structured polyglycolic acid.

FIGS. 15A, 15B, 15C and 15D are images of the surface morphology ofuntreated polypropylene, nano-structured polypropylene, untreatedpolypropylene mesh fibers and nano-structured polypropylene mesh fibers.

FIGS. 16A and 16B are images of the surface morphology of untreatedpolyvinyl chloride and nano-structured polyvinyl chloride.

FIGS. 17A, 17B, 17C and 17D are images of the surface morphology ofuntreated polyethylene and nano-structured polyvinyl chloride.

FIGS. 18A, 18B, 18C, 18D, 18E and 18F are AFM images of a siliconesubstrate with various nano-structured surfaces.

FIG. 19 is a depiction of the surface morphology of an untreated medicaldevice and a medical device have a surface treated to produce anano-structured surface.

FIG. 20 are SEM images of untreated polyvinyl chloride and polyvinylchloride treated with R. arrhisus to produce a nano-structured surface.

FIG. 21 are AFM images of untreated polyvinyl chloride and polyvinylchloride treated with R. arrhisus to produce a nano-structured surface.

FIG. 22 are XPS graphs of untreated polyvinyl chloride (top) andpolyvinyl chloride treated with R. arrhisus to produce a nano-structuredsurface (bottom) showing carbon and chlorine peaks indicating that thecreation of a nano-structured surface did not alter material chemistry.

FIG. 23 is a graph of colony forming units (CFU) of Pseudomonasaeruginosa removed using various methods from untreated polyvinylchloride and polyvinyl chloride treated with R. arrhisus to produce anano-structured surface.

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Embodiments of the present invention are based on the discovery thatsurfaces having a nanometer scale geometry, architecture or structuredesirably reduce adhesion of bacteria. Since adhesion of bacteria to thesurface is reduced, proliferation of bacterial cells is reducedaccording to embodiments of the present invention and the risk ofbacterial infection or illness is also reduced according to embodimentsof the present invention.

Bacteria within the scope of the present disclosure includesStaphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa,MRSA, E. coli, candida (yeast), Streptococcus pneumoniae, Neisseriameningitides, Haemophilus influenzae, Streptococcus agalactiae, Listeriamonocytogenes, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionellapneumophila, Mycobacterium, tuberculosis, Streptococcus pyogenes,Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum,Ureaplasma urealyticum, Haemophilus ducreyi, Helicobacter pylori,Campylobacter jejuni, Salmonella, Shigella, Clostridium,Enterobacteriaceae, Staphylococcus saprophyticus and the like. The abovelist is intended to be exemplary and not exhaustive. One of skill in theart will readily be able to identify additional bacteria within thescope of the present disclosure.

Embodiments within the scope of the present invention include substratesthat are capable of supporting a nanometer scale geometry, architectureor structure, also referred to herein as “roughness.” The surface of thesubstrates themselves can be altered to remove material from the surfacethereby creating a nanometer scale roughness. Alternatively, materialmay be added to the surface to create a nanometer scale roughness. Stillfurther, the substrate itself can be manufactured such as with a mold tohave a surface with a nanometer scale roughness. With each of the aboveembodiments, the result is a substrate with a surface with a nanometerscale roughness.

Substrates within the scope of the present invention can be fashionedfrom any material that can support or be altered to provide a nanometerscale roughness. In a particular embodiment, substrates can include anymaterials susceptible of being degraded, etched or otherwise altered bynano-roughing agents.

Suitable materials include metals, polymers, ceramics, and compositesthereof and the like. Metals according to the invention includetitanium, aluminum, platinum, niobium, tantalum, tin, nickel, cobalt,chromium, molybdenum, stainless steel, nitinol, Ti6A14V, SiN, CoCrMo,and alloys thereof and the like. The above list is intended to beexemplary and not exhaustive. One of skill in the art will readily beable to identify additional metals and metal alloys within the scope ofthe present disclosure. Polymers according to the invention includepolyvinyl chloride, silicone, polyurethane, polycaprolactone,poly-lactic-co-glycolic acid, poly-lactic acid, poly-glycolic acid,polyethylene, polyethylene glycol, polydimethylsiloxane, polyacrylamide,polypropylene, polystyrene, polyether ether ketone (PEEK), ultra highmolecular weight polyethylene (UHMWPE), hydrogels, and compositesthereof and the like. The above list is intended to be exemplary and notexhaustive. One of skill in the art will readily be able to identifyadditional polymers within the scope of the present disclosure. Ceramicsaccording to the invention include alumina, titania, hydroxyapatite,silica, calcium phosphates, bone cements, and composites thereof and thelike. The above list is intended to be exemplary and not exhaustive. Oneof skill in the art will readily be able to identify additional ceramicmaterials within the scope of the present disclosure. According to aparticular embodiment, polymers that are used to fashion devices to beinserted or implanted into an individual, such as poly vinyl chlorideand silicon based polymers such as silicones, are useful in the presentinvention.

Devices within the scope of the present invention that benefit from areduction of adhesion of bacteria are devices that are intended to beinserted or implanted within an individual. Additional devices includethose where food is to be prepared or materials or devices intended tobe inserted or implanted are manufactured or staged prior to use. Suchdevices are commonly found in food processing rooms, kitchens,manufacturing clean rooms, operating rooms and the like. Specificexamples of devices intended to be inserted or implanted within anindividual include tubes, such as endotracheal tubes, central venous,arterial, and urinary catheters, stents, dialysis tubing, catheters,orthopedic and dental implants, vascular implants, pacemaker leads,neural probes, neural catheters, wound healing devices, skin patches,hernia meshes, spinal implants and the like. Specific examples ofdevices where food is to be prepared or materials or devices intended tobe inserted or implanted are manufactured or staged prior to use such asin an operating room, include tabletops, countertops, trays, plasticcutting boards and the like.

According to certain aspects of the present invention, the surface ofthe substrate can be altered to produce a nanometer scale surfaceroughness using one or more nano-roughing agents whether physical orchemical. In this manner, a portion of the substrate surface can beremoved by action of a nano-roughing agent. Mechanisms for removingmaterial from a substrate surface include abraiding, degrading,dissolution, etching and the like to produce a nanometer scale surfaceroughness. Particular methods include contacting the surface of thesubstrate with a device that will remove material from the substratesurface, such as by friction or abrasion. Alternatively, a liquid orgaseous material can be applied to the surface of the substrate todegrade, dissolve or etch away material from the surface of thesubstrate to produce a nanometer scale surface roughness. Suchtreatments are referred to as chemical treatments and include liquid orgaseous materials such as acids, bases, lipases, dichloroethylene andxylene and the like. Surfaces produced by the above methods reduce thegrowth of bacteria thereon. Exemplary nano-roughing agents include oneor more of an acid, a base, an alcohol, a peroxide, isoamyl acetate,dichloromethane, isoamyl acetate with zinc, dichloromethane with zinc,acetic acid, sulfuric acid, nitric acid, perchloric acid, phosphoricacid, hydrochloric acid, chloroform, acetone, ethanol, ammonia, sodiumhydroxide, potassium hydroxide, ammonium hydroxide, ammonium fluoride,hydrofluoric acid, triflic acid, hydrogen peroxide, dichloroethylene,xylene and the like and the bacterial lipases previously mentioned. Oneof skill in the art will readily identify additional nano-roughingagents based on the present disclosure.

According to an exemplary embodiment of the present invention, bacteriallipase solutions are used to produce a nanometer scale surface roughnesson a substrate. According to this aspect, substrate surfaces arecontacted with lipases, for example from C. cilindracea and R. arrhisus,in a manner to cause enzymatic degradation of the substrate andnanometer scale features on the surface of the substrate. In thismanner, a method is provided to create a nanometer scale surfaceroughness having antibacterial properties by contacting the surface of asubstrate with one or more lipases for a period of time to allowenzymatic degradation of surface materials thereby creating nanometerscale features on the surface of the substrate. In addition to lipasesfrom C. cilindracea and R. arrhisus, other useful lipases include thosefrom Candida rugosa, Thermus thermophilus, Candida Antarctica,Aspergillus niger, Aspergillus oryzae, Aspergillus sp, Burkholderia sp,Candida utilis, Chromobacterium viscosum, Mucor javanicus, Penicilliumroqueforti, Pseudomonas cepacia and the like. Other useful lipases andetchants include phospholipases, sphingomyelinases, hepatic lipase,endothelial lipase, lipoprotein lipase, bile salt dependent lipase,pancreatic lipase, lysosomal lipase, hormone-sensitive lipase, gastriclipase, pancreatic lipase related protein 2, pancreatic lipase relatedprotein 1, lingual lipase and the like.

According to certain aspects of the present invention, the surface of asubstrate can be altered in a manner such that material is added to thesubstrate surface to produce a nanometer scale surface roughness.Material, either the same or different from the material of thesubstrate surface can be deposited on the surface of the substrate usingdeposition methods such as vapor deposition well known to those of skillin the art.

According to certain other aspects of the present invention, a nanometerscale surface roughness can be created during the manufacture of thesubstrate surface such as by use of a mold or other device such as astamp that can leave a nanometer scale surface roughness imprint ontothe surface of the substrate.

According to one aspect of the present invention, the surface of themedical device is modified to include a nanostructured outer surface,that is one characterized by the presence of physical structures havingnanometer scale dimensions such as height and/or width. A medical devicewith such a surface according to the present invention limits, inhibitsprevents and/or reduces bacterial adhesion as compared to a devicewithout the nanostructured outer surface.

Examples of such medical devices are those utilizing polyvinyl chloride(PVC) or silicon polymer tubes, such as endotracheal tubes and centralvenous, arterial, and urinary catheters. Certain embodiments of thepresent invention are directed to nanostructures on the surface of amedical device that limit adhesion of infection-causing bacteria to thesurface of the medical device. According to certain aspects, thenanostructures provide a surface chemistry, surface geometry, surfacefree energy or condition which limits bacterial adhesion, proliferationand/or differentiation. The nanostructures can take the appearance ofetching on the surface of the medical device, for example in thegeometry or structural features of lines, points, hills, mounds,valleys, slopes and the like and distances between such geometries andstructural features of various nanometer dimensions such as heightand/or width and or length and/or depth having dimensions in the rangefrom about 1 nanometer to about 1000 nanometers. For example nanoscalefeatures within the scope of the present invention include those havingdimensions between about 10 nanometers to about 900 nanometers, about100 nanometers to about 500 nanometers, about 1 nanometer to about 100nanometers, about 10 nanometers to about 50 nanometers, about 1nanometer to about 10 nanometers, about 1 nanometer to about 5nanometers, about 10 nanometers to about 100 nanometers, and any rangesin between the above ranges. In one embodiment the lines of etching arespaced about 600 nm from each other. In another embodiment the lines ofetching are spaced about 500 nm from each other.

Embodiments of the present invention are also directed to methods ofobtaining nanostructured medical devices. According to this aspect ofthe present invention, one or more nano-roughing agents such asbacterial lipases are selected that will chemically or enzymaticallydegrade the surface of a medical device when contacted to the surfacefor a period of time and at a sufficient concentration. One of skill inthe art will readily recognize concentrations, time periods andtemperatures within the scope of the invention based on the benefit ofthe disclosure herein. According to one embodiment, the bacterial lipaseis C. cilindracea. According to another embodiment, the bacterial lipaseis R. arrhisus.

Embodiments of the present invention are also directed to methods oflimiting bacterial adhesion to the surface of a medical device.According to this aspect of the present invention, the nanostructuresalter the surface of the medical device. The nanostructured surfaceprovides a different surface roughness than a nanosmooth surface.Without wishing to be bound by theory, it is believed that thenanostructured surface inhibits or prevents the adhesion of bacterialcells to the surface. According to additional embodiments, ananostructured surface creates a surface charge or hydrophobicity thatinhibits, reduces, limits and/or prevents adhesion and or growth ofbacterial cells to or on the surface.

Embodiments of the present invention are directed to methods ofinhibiting the rate of growth of bacteria over a prolonged period oftime. According to one embodiment, a device is provided with or alteredto include a nanostructured surface, the nanostructured surface iscontacted with bacteria, and the rate of bacterial growth is inhibitedor reduced over a prolonged period of time. According to certainembodiments, the rate of bacterial growth is reduced over a prolongedperiod of time including hours, such as about 4 hours, about 12 hours,about 24 hours, about 72 hours, etc., over a period of days, such asabout 1 day, about 2 days, about 3 days, about 4, about 5 days, about 6days, about 7 days etc., over a period of weeks, such as 1 week, 2weeks, 3 weeks, 4 weeks, 5, weeks, etc., over a period of months, suchas about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, to about 12 months and overa period of years, such as 1, 2, 3, 4, 5 years etc. It is to beunderstood that combinations of the above time periods are within thescope of the present invention such as the prolonged period of timecould be one week and 3 days, one month and two weeks and four days, oneyear and three months and one week and two days, etc.

It is to be further understood that embodiments of the present inventioninclude methods of permanently rendering a surface of a substrateresistant to bacterial growth. Such embodiments include the method ofproviding a substrate with a permanent nanostructured surface,contacting the surface with bacteria, and reducing the adherence and/orgrowth, and/or proliferation and/or accumulation of bacteria on thesurface of the substrate. According to this aspect of the presentinvention, the methods above for creating a nanostructured surface areperformed on a substrate material that retains the nanostructuredsurface features under normal wear and tear and common environmentaland/or physioloigical conditions. For example, creating a nanostructuredsurface on a PVC material according to the methods describe herein isconsidered permanent insofar as the nanostructured surface will remainunaltered at temperatures and environmental conditions which do notcause the PVC material to change its structure. One such set ofconditions is physiological conditions and common room temperatureenvironmental conditions. Conditions which could cause the PVC materialto alter its structure include coming in contact with heat sufficient tomelt and/or destroy the PVC and/or solvents which could dissolve the PVCmaterial. Under this embodiment, the term “permanent” includes theuseful life of the substrate including the nanostructured surface. Solong as the nanostructured surface is capable of coming into contactwith bacteria, and the surface retains its nanometer scale surfacestructural features, the surface is permanently rendered resistant tobacterial growth.

An additional embodiment of the present invention includes a method ofimproving resistance of a substrate to bacterial growth includingproviding a substrate surface with features having nanometer scaledimensions, i.e. a nanostructured surface, contacting the surface withbacteria, reducing the growth of bacteria, removing bacterial growth,and repeating the steps of contacting, reducing and removing in whole orin part any numbered of desired times. According to this embodiment, thesubstrate having the nanostructured surface is reusable in methods ofreducing growth of bacteria or otherwise reducing the rate of growth ofbacteria. The step of removing is accomplished by common wiping orcleaning or sterilizing techniques known to those of skill in the art.

It is to be understood that the embodiments of the present inventionwhich have been described are merely illustrative of some of theapplications of the principles of the present invention. Numerousmodifications may be made by those skilled in the art based upon theteachings presented herein without departing from the true spirit andscope of the invention. The contents of all references, patents andpublished patent applications cited throughout this application arehereby incorporated by reference in their entirety for all purposes.

The following examples are set forth as being representative of thepresent invention. These examples are not to be construed as limitingthe scope of the invention as these and other equivalent embodimentswill be apparent in view of the present disclosure, figures, andaccompanying claims.

EXAMPLES

The following examples are specific embodiments of the present inventionbut are not intended to limit it.

Example 1 Formation of Nanostructured Surfaces on Endotracheal TubesUsing R. Arrhisus

0.1% and 0.5% mass solutions of Rhisopus arrhisus were each prepared inpotassium phosphate buffer at a pH of 7.4. 10 mL of each solution wasthen placed in a glass Petri dish with a sample of a polyvinyl chloride(PVC) endotracheal tube (1 cm×1 cm). The PVC sample was left at 37° C.for 24 hours. The sample was then removed, washed with distilled water,and returned to the Petri dish with fresh solution for another 24 hours.The enzymatic degradation of the PVC was measured for 48 hours. Theactivity of R. arrhisus was measured to be 10.5 U/g. One unit is definedto be the amount of enzyme that catalyzed the release of 1 μmol of oleicacid per minute.

The samples were observed at the 24 hour and 48 hour mark using ascanning electron microscope (SEM). As a control, SEM micrographs ofuntreated PVC samples were taken at magnifications of 1 k (FIG. 1A) and42 k (FIG. 1B). After 24 hours of soaking in a 0.5% solution of R.arrhisus, nano features could be seen on the treated PVC sample as shownin FIG. 2A at magnification of 20 k. After 48 hours of soaking in a 0.1%R. arrhisus solution, nano features could be seen on the treated PVCsample as shown in FIG. 4A (10 k magnification) and FIG. 4B (42 kmagnification). After 48 hours of soaking in a 0.5% R. arrhisussolution, nano features could be seen on the treated PVC sample as shownin FIG. 4C (20 k magnification) and FIG. 4D (42 k magnification).Visible nano features with clear etching of the surface wasdemonstrated. The etching produced substantially uniform lines spacedabout 500 nm apart. In a separate experiment, SEM micrographs were takenof untreated PVC and PVC treated with R. arrhisus at 0.5% for 72 hoursshowing the scale of features produced by the treatment. As shown inFIGS. 6A and 6B, the treatment with R. arrhisus produced nanometer scalefeatures.

Example 2 Formation of Nanostructured Surfaces on endotracheal EubesUsing C. Cilindracea

0.1% and 0.5% mass solutions of Candida cilindracea were each preparedin potassium phosphate buffer at a pH of 7.4. 10 mL of each solution wasthen placed in a glass Petri dish with a sample of a polyvinyl chloride(PVC) endotracheal tube (1 cm×1 cm). The PVC sample was left at 37° C.for 24 hours. The sample was then removed, washed with distilled water,and returned to the Petri dish with fresh solution for another 24 hours.The enzymatic degradation of the PVC was measured for 48 hours. Theactivity of C. cilindracea was measured to be 7.29 U/g.

The samples were observed at the 24 hour and 48 hour mark using ascanning electron microscope (SEM). After 24 hours of soaking in a 0.5%solution of C. cilindracea, nano features could be seen on the treatedPVC sample as shown in FIG. 2B at magnification of 20 k. After 48 hoursof soaking in a 0.1% C. cilindracea solution, nano features could beseen on the treated PVC sample as shown in FIG. 3a (5 k magnification)and FIG. 3B (42 k magnification). Visible nano features with clearetching of the surface was demonstrated. The etching producedsubstantially uniform lines spaced about 600 nm apart. As shown in theSEMs, the features having nanometer scale dimensions have the shapes oflines, dots, spots, hills, points, mounds, valleys, and slopes.According to one embodiment shown, the features are in the shape ofelongated features such as lines, broken lines, spots in series forminga lines, wherein one or more of the elongated features are substantiallyparallel to one another and are of a substantially similar height andwidth.

Example 3 Decreased Staphylococcus Aureus Growth on NanostructuredSurfaces

Staphylococcus aureus was grown on nanostructured PVC that had beentreated with R. arrhisus and C. cilindracea in the manner describedabove, as well as on a control with no etching. Bacteria were culturedin trypic soy broth under standard biological conditions, specificallyunder a 37° C., humidified, 5%CO₂/95% air environment. The samples werestained with Crystal Violet and the growth of bacteria was measured at 4hours, 12 hours, 24 hours, and 72 hours. A substantial decrease inbacterial growth and/or reduction in the rate of bacterial growth orproliferation was seen between the untreated sample and the treatedsamples over time. According to methods of the present invention, asubstantial reduction in growth of bacteria is achieved after 72 hours.Given the permanent nanostructured surface of the substrate used in thisexperiment and the demonstrated reduction in bacterial growth, one ofskill will understand that the reduction in the growth or rate of growthwill continue even longer and for the useful life of the substrate. Oneof skill will further understand that the surface of the substrate hasbeen rendered permanently resistant to bacterial growth, and that thecapability of the substrate to resist bacterial growth is enhanced byremoving bacteria that have accumulated on the surface of the substrate.One of skill will further understand based on this example that otheretchants as described above can be used to produce a surface of asubstrate having nanometer scale features. Surfaces composed of one ormore materials as described above other than PVC are useful in thepresent invention. The substrate having the nanosurface thereon is anyof the substrates described above where, for example, reduced orinhibited bacterial growth is desired. According to the presentinvention, substrates such as endotracheal tubes, can be altered toinclude a surface having nanometer scale features and the endotrachealtube can be used in the methods of reducing growth or rate of growth ofbacteria or otherwise reducing the risk of bacterial infection thatoften accompanies introducing such a substrate into a patent.

Example 4 Formation of Nano-Structured Surface on Polypropylene

Polypropylene substrate samples were treated in either isoamyl acetateor dichloromethane for 42 hours using a method similar to that describedin Example 1. For some of the samples, ZnCl was added to either theisoamyl acetate or dichloromethane to introduce Zn particles onto thesubstrate. Upon complete of the treatment, the samples were removed andrinsed three times in distilled water.

Example 5 Decreased Inflammatory Cell Functions

Macrophages (ATCC) were seeded at 2500 cells/cm² onto the substrates ofinterest in standard cell culture media for 4 hours and 1 and 5 daysunder standard cell culture conditions. At the end of the prescribedtime period, adherent cells were determine using standard MTT assays.Experiments were repeated in triplicate at least three times each. Asshown in FIG. 7, nanostructured polypropylene (withoutlipopolysaccharide) exhibited less macrophage density.

Macrophage experiments were conducted as described above. As shown inFIG. 8, nanostructured polypropylene (with lipopolysaccharide) exhibitedless macrophage density.

Example 6 Decreased Bacteria Functions

As shown in FIGS. 9A and 9B, a nanostructured surface of poly etherether ketone (PEEK) demonstrated antibacterial properties against Staph.epidermidis using a method similar to that described in Example 3.

As shown in FIG. 10, a nanostructured surface of PVC demonstratedantibacterial properties against Staphylococcus aureus using substratestreated with either Rhisopus arrhisus or Candida cilindracea.

As shown in FIG. 11, a nanostructured surface of polypropylenedemonstrated antibacterial properties against Staph. epidermidiscolonization.

Example 7 Enhanced Osteoblast Calcium Deposition on Nano-Structured PEEK

Osteoblasts (ATCC) were seeded on the substrates of interest at 40,000cells/cm² and were cultured in standard cell culture media for 1, 2, and3 weeks under standard cell culture conditions with the media changedevery other day. At the end of the prescribed time period, samples wererinsed in saline solution and soaked in HCl to dissolve calciumdeposited by the osteoblasts in the extracellular matrix. The calciumcontaining supernatants were then analyzed using the Calcium Assay(Sigma). Experiments were completed in triplicate at least three timeseach. As shown in FIG. 12, nano-structured PEEK demonstrated enhancedosteoblast calcium deposition.

Example 8 Nanostructured Surface Materials

As shown in the SEM images of FIGS. 13A and 13B, the control (untreated)surface of polyetherether ketone (FIG. 13A) is smooth relative to thenano-rough surface morphology of polyetherether ketone (FIG. 13B)treated using a protocol similar to that described in Example 1.

As shown in the SEM image of FIGS. 14A and 14B, the control (untreated)surface of polyglycolic acid (FIG. 14A) is smooth relative to thenano-rough surface morphology of polyglycolic acid (FIG. 14B) treatedusing a protocol similar to that described in Example 1.

As shown in the SEM image of FIGS. 15A, 15B, 15C and 15D, the control(untreated) surface of polypropylene (FIG. 15A) is smooth relative tothe nano-rough surface morphology of polypropylene (FIG. 15B) treatedusing a protocol similar to that described in Example 1. As also shownin the SEM image, the control (untreated) surface of polypropylene meshfibers (FIG. 15C) is smooth relative to the nano-rough surfacemorphology of polypropylene mesh fibers (FIG. 15D) treated using aprotocol similar to that described in Example 1.

As shown in the SEM image of FIGS. 16A and 16B, the control (untreated)surface of polyvinyl chloride (FIG. 16A) is smooth relative to thenano-rough surface morphology of polyvinyl chloride (FIG. 16B) treatedusing a protocol similar to that described in Example 1.

As shown in the SEM image of FIGS. 17A, 17B, 17C and 17D, the control(untreated) surface of polyethylene (FIG. 17A) and (FIG. 17C) is smoothrelative to the nano-rough surface morphology of polyethylene (FIG. 17B)and (FIG. 17D) treated using a protocol similar to that described inExample 1.

As shown in the AFM images of FIGS. 18A, 18B, 18C, 18D, 18E and 18F,silicone has been rendered nano-structured according to the methods ofthe present disclosure using a protocol similar to that described inExample 1. The first image is a control with no surface treatment andaccordingly no nanoscale roughness. The remaining images with surfacetreatment show various nanoscale surface features.

Example 9 Nanostructured Medical Devices

FIG. 19 is an image of the relative surface morphology of the untreatedtitania surface of a medical device and the titania surface of a medicaldevice treated to have nanoscale surface features through the compactionof titania nanoparticles. Specifically, titania nanoparticles (23 nmdiameter) obtained from Nanophase Technologies, Inc. were pressed in aserial dye at 1 GPa of pressure over 10 minutes to form a compact withnanoscale features (bottom). The top image is the roughness of astandard hip implant which shows a lack of nanometer surface features.The nanostructured surface inhibits the attachment, growth and/ordifferentiation of bacterial cells.

Example 10 Decreased Pseudomonas Aeruginosa Growth on NanostructuredSurfaces

The effect of a nanostructured surface on the growth of Pseudomonasaeruginosa was determined as follows. Commercially available Sheridan®6.0 mm ID, uncuffed endotracheal tubes (ETTs) (Hudson RIC, Temecula,Calif.) were cut vertically into 0.6 cm by 0.3 cm segments using arectangular hole punch. To create a nanorough (Nano-R) topography on thePVC, a lipase from Rhizopus arrhizus (Sigma-Aldrich, St. Louis, Mo.) wasused at a 0.1% concentration in a 1M potassium phosphate buffer. The PVCsamples were soaked in the Rhizopus arrhizus solution at 37° C., 200 rpmfor 24 h. After 24 h, the solution was replaced with fresh solution andthe samples were soaked for an additional 24 h. Upon completion of theenzymatic treatment, the Nano-R samples were removed and rinsed threetimes with double distilled water. The samples were then removed anddried overnight at room temperature. When the Nano-R samples werecompletely dry, they were sterilized using ethylene oxide gas. UntreatedPVC samples were also sterilized using ethylene oxide gas prior toexperimentation.

Surface topography of the material was visualized using a scanningelectron microscope (LEO 1530VP FE-4800 Field-Emission SEM, Carl ZeissSMT, Inc. Peabody, Mass.) according to standard operating procedure.Samples were mounted on aluminum stubs, sputter coated to provide aconductive gold/palladium coating with a thickness of 90 A, and imagedwith an accelerating voltage of 0.5 to 2 kV. As shown in FIG. 20, SEMimages of untreated PVC at 5K× magnification (A) and 50K× magnification(B) revealed a surface topography that is smooth at the nanoscale.Images of Nano-R surfaces at 5K× magnification (C) and 50K×magnification (D) show a nanorough topography revealing surfacedegradation of the PVC.

The topography of the PVC substrates were evaluated with atomic forcemicroscopy (AFM: XE-100, Park Systems Inc, Santa Clara, Calif.) undertapping mode using a 10 nm AFM tip (PPP-NCHR, Park Systems Inc, SantaClara, Calif.) with a scan rate of 0.5 Hz. As shown in FIG. 21, AFMimages and results for untreated PVC (top) and Nano-R PVC (bottom)indicated RMS values of 2.155±0.7955 nm for untreated PVC and45.298±18.785 nm for Nano-R PVC.

To compare the surface chemistry of the treated and untreated PVCsamples, X-ray photoelectron spectroscopy (XPS) analysis was used. Abrief sputter cleaning was performed to eliminate surface contamination.Parameters of the XPS system (5500 Multitechnique Surface Analyzer,Perkin Elmer, Waltham, Mass.) were adjusted to analyze the top 50 A ofthe material surface over a circular area with a diameter of 1.1 mm.Acquired data was processed with appropriate software (PC Access ESCAV7.2C, Physical Electronics, Chanhassen, Minn.). As shown in FIG. 22,XPS data of untreated PVC (top) and Nano-R PVC (bottom) revealed carbonand chlorine peaks indicative of PVC. Matching peaks indicated that thecreation of Nano-R topography did not alter material chemistry.

Pseudomonas aeruginosa (ATCC 25668, American Type Culture Collection,Manassas, Va.) was hydrated and streaked for isolation on a tryptic soyagar plate. Following growth, a single isolated colony was selected andused to inoculate 5 ml of tryptic soy broth (TSB). The bacteria culturewas grown on an incubator shaker for 18 hours at 37° C., 200 rpm. Thebacteria suspension was diluted 1:30, and 150 μl of the bacteria culturewas added to a single well of a round bottom, non-tissue treated 96 wellplate containing either a Nano-R or an untreated PVC sample. The platewas then placed in a stationary incubator at 37° C. with 5% carbondioxide. After 24 hours, the plate was removed, and the excess media wascarefully aspirated. Media residue and non-adherent bacteria werecarefully rinsed from sample surfaces with phosphate buffered saline(PBS). Following the removal of the PBS, the PVC samples were extractedfrom the well plates using a pair of sterile forceps. Samples weregently removed and an effort was made to minimize forcep contact thatcould disrupt biofilms on the surfaces. Each sample was then placed intoa 20 mL disposable scintillation vial containing 2 ml of TSB for furthermechanical stimulation to remove biofilm. An additional control vialreceived no additives or mechanical stimulation. Samples were kept atroom temperature from the time of sample transfer to the scintillationvials until processing within 30 min according to one of the fourmethods described below.

Biofilm Removal Process 1: The vial containing the PVC sample wasvortexed for 1 min at 3000 rpm. The degree of turbulence in the media inthe scintillation vial depended on the angle at which the vial was heldonto the vortex. The firmness with which the vial was held also affectedthe apparent degree of turbulence. For consistency, the vials were heldin a vertical position with a slightly softened grip. Proper vortexingwas indicated by the appearance of media swirling around thecircumference of the vial from top to bottom.

Biofilm Removal Process 2: The vial containing the PVC sample was placedin a tabletop ultrasonic cleaner (B3500A, VWR International, Batavia,Ill.) and sonicated for 10 min at the highest setting. The output of theultrasonic cleaner, which contained 5.7 L of water, was 135 W at afrequency of 42 kHz.

Biofilm Removal Process 3: The vial containing the PVC sample wasvortexed for 1 min at 3000 rpm as described above, and then sonicatedfor 10 min on the highest setting as described above.

Biofilm Removal Process 4: The vial containing the PVC sample includesTWEEN 80 in the TSB (5% by volume). The vial was vortexed for 1 min at3000 rpm.

Following each of the four treatments described above, the supernatantmedia was serially diluted and plated on tryptic soy agar plates toquantify the number of viable cells removed from the surfaces. For allexperimental and control groups, the 10⁻⁵ dilution plating providedcolony numbers in a countable range. Colony forming units (CFU) ofPseudomonas aeruginosa were counted via a spread plate technique afteran overnight incubation. As shown in FIG. 23, all methods of removal(with the exception of vortexing plus ultrasound [V+US]) showed agreater Pseudomonas population on untreated samples compared to Nano-RPVC, with vortexing providing high CFU yield. Values are mean±SEM; N=3;*p<0.05 (compared to untreated samples under same conditions),^(#)p<0.05 (for both untreated and Nano-R compared to control). Thisdemonstrates a reduced presence of bacteria on the Nano-R PVC achievedwithout the use of antimicrobial agents. Numerical data were analyzedfor significance using the student's t-test (N=3). Experiments wereperformed in triplicate. Values are reported as the mean±SEM. Thethreshold for significance was set at p<0.05.

Given the benefit of the above disclosure and description of exemplaryembodiments, it will be apparent to those skilled in the art thatnumerous alternative and different embodiments are possible in keepingwith the general principles of the invention disclosed here. Thoseskilled in this art will recognize that all such various modificationsand alternative embodiments are within the true scope and spirit of theinvention. While the invention has been illustrated and described indetail in the drawings and foregoing description, such illustration anddescription is to be considered as exemplary and not restrictive incharacter, it being understood that, only the preferred embodiments havebeen shown and described and that all changes and modifications thatcome within the spirit of the invention are desired to be protected. Theappended claims are intended to cover all such modifications andalternative embodiments. It should be understood that the use of asingular indefinite or definite article (e.g., “a,” “an,” “the,” etc.)in this disclosure and in the following claims follows the traditionalapproach in patents of meaning “at least one” unless in a particularinstance it is clear from context that the term is intended in thatparticular instance to mean specifically one and only one. Likewise, theterm “comprising” is open ended, not excluding additional items,features, components, etc. References identified herein are expresslyincorporated herein by reference in their entireties unless otherwiseindicate.

What is claimed is:
 1. A method of reducing growth of bacteria on asurface of a substrate comprising altering the surface of the substrateto produce a nanometer scale surface geometry by contacting the surfaceof the substrate with a solution of a nano-roughing agent to produce ananometer scale surface geometry on the surface of the substrate,contacting the surface with bacteria and inhibiting growth of thebacteria.
 2. The method of claim 1 wherein the nano-roughing agent isone or more of an acid, a base, a bacterial lipase, an alcohol, aperoxide, isoamyl acetate, dichloromethane, isoamyl acetate with zinc,dichloromethane with zinc, acetic acid, sulfuric acid, nitric acid,perchloric acid, phosphoric acid, hydrochloric acid, chloroform,acetone, ethanol, ammonia, sodium hydroxide, potassium hydroxide,ammonium hydroxide, ammonium fluoride, hydrofluoric acid, triflic acid,hydrogen peroxide, dichloroethylene, or xylene.
 3. The method of claim 1wherein the nano-roughing agent is one or more of phospholipases,sphingomyelinases, hepatic lipase, endothelial lipase, lipoproteinlipase, bile salt dependent lipase, pancreatic lipase, lysosomal lipase,hormone-sensitive lipase, gastric lipase, pancreatic lipase relatedprotein 2, pancreatic lipase related protein 1, lingual lipase, andbacterial lipase produced from one or more of Rhisopus arrhisus, Candidacilindracea, Candida rugosa, Thermus thermophilus, Candida Antarctica,Aspergillus niger, Aspergillus oryzae, Aspergillus sp, Burkholderia sp,Candida utilis, Chromobacterium viscosum, Mucor javanicus, Penicilliumroqueforti, or Pseudomonas cepacia.
 4. The method of claim 1 wherein thesurface of the substrate is contacted with a solution of Rhisopusarrhisus or Candida cilindracea.
 5. The method of claim 1 wherein thesurface of the substrate includes one or more of a metal, a polymer or aceramic.
 6. The method of claim 5 wherein the metal is one or more oftitanium, aluminum, platinum, niobium, tantalum, tin, nickel, cobalt,chromium, molybdenum, stainless steel, nitinol, Ti6A14V, SiN, CoCrMo, ormixtures or alloys thereof.
 7. The method of claim 5 wherein the polymeris one or more of polyvinyl chloride, silicone, polyurethane,polycaprolactone, poly-lactic-co-glycolic acid, poly-lactic acid,poly-glycolic acid, polyethylene, polyethylene glycol,polydimethylsiloxane, polyacrylamide, polypropylene, polystyrene,polyether ether ketone (PEEK), ultra high molecular weight polyethylene(UHMWPE),or hydrogels, or composites thereof
 8. The method of claim 5wherein the ceramic is one or more of alumina, titania, hydroxyapatite,silica, calcium phosphates, or bone cements, or composites thereof. 9.The method of claim 1 wherein the substrate is an endotracheal tube 10.The method of claim 1 wherein the bacteria is one or more ofStaphylococcus aureus, Staphylococcus epidermis, Pseudomonas aeruginosa,MRSA, E. coli, candida (yeast), Streptococcus pneumoniae, Neisseriameningitides, Haemophilus influenzae, Streptococcus agalactiae, Listeriamonocytogenes, Mycoplasma pneumoniae, Chlamydia pneumoniae, Legionellapneumophila, Mycobacterium, tuberculosis, Streptococcus pyogenes,Chlamydia trachomatis, Neisseria gonorrhoeae, Treponema pallidum,Ureaplasma urealyticum, Haemophilus ducreyi, Helicobacter pylori,Campylobacter jejuni, Salmonella, Shigella, Clostridium,Enterobacteriaceae, or Staphylococcus saprophyticus
 11. A method ofinhibiting growth of bacteria on the surface of a substrate comprisingproviding the surface of the substrate with a nanometer scale surfacegeometry, contacting the surface with bacteria, and inhibiting adherenceof the bacteria to the surface.
 12. A method of inhibiting growth ofbacteria on the surface of a substrate comprising providing the surfaceof the substrate with a nanometer scale surface geometry, contacting thesurface with bacteria, and inhibiting growth of the bacteria on thesurface.
 13. A method of reducing bacterial infection in an animalincluding a human comprising inserting into the animal a substratehaving a surface with a nanometer scale surface geometry, contacting thesurface with bacteria, and inhibiting growth of the bacteria on thesurface.
 14. A method of reducing bacterial infection associated with anendotracheal tube in an animal including a human comprising insertinginto the animal the endotracheal tube having a surface with a nanometerscale surface geometry, contacting the surface with bacteria, andinhibiting growth of the bacteria on the surface.
 15. An anti-bacterialsubstrate comprising a substrate and a substrate surface having ananometer scale surface geometry sufficient to inhibit adherence ofbacterial cells to the substrate surface.